The action of insulin is mediated through the insulin receptor (IR), an integral membrane glycoprotein. To transduce a signal, the receptor phosphorylates the tyrosine residues of insulin receptor substrate I (IRS- I). IRS-I binds to the SH2 domains in various signal transduction proteins and triggers a phosphorylation cascade, thereby altering the cellular function. To study the regulation of the human insulin receptor gene, the cis-elements and trans-acting factors that determine transcription have previously been identified. Aim 1 is to study the mechanism by which Rb stimulates hIR gene expression. The action of retinoblastoma gene product (Rb), a regulator of cellular proliferation, is mediated through clustered Spl binding sites. It is hypothesized that Rb might induce hIR expression by sequestering a suppressor protein. The mechanism by which Rb regulates hIR gene expression will be determined and the factor which mediates Rb induction of transcription of the hIR gene will be cloned. Aim 2 is to clone and characterize IRNF-I and IRNF-II, two nuclear factors that are important for insulin receptor gene transcription. Aim 3 is to examine the enhancement of hIR gene expression during adipocyte differentiation. The expression of hIR is highly regulated during adipocyte differentiation. The possible effects of PPARgamma2 and C/EBPalpha on this process will be examined and the mechanisms of action determined.